The current invention relates to methods for inhibiting and/or reversing tau filament formation or polymerization. This invention also relates to methods for treating certain neurological disorders in vivo by administering pharmaceutical compositions which inhibit and/or reverse tau filament formation or polymerization.
The microtubule-associated protein tau is a soluble cytosolic protein that is believed to contribute to the maintenance of the cytoskeleton (Johnson et al., Alzheimer""s Disease Review 3: 125 (1998); Buee et al., Brain Research Reviews 33:95 (2000)). However, in many disease states, tau protein is induced by unknown cellular conditions to self-associate into filamentous structures (Spillantini et al., Trends Neurosci. 21: 428 (1998)). These filamentous forms of tau can be found in such varied neurodegenerative disorders as Alzheimer""s disease (AD) (Wood et al., Proc. Natl. Acad. Sci. USA 83: 4040 (1986); Kosik et al., Proc. Natl. Acad. Sci. U.S.A 83: 4044 (1986); Grundke-Iqbal et al., J. Biol. Chem. 261: 6084 (1986)), corticobasal degeneration (CBD) (Feany et al., Am. J. Pathol. 146: 1388 (1995)), progressive supranuclear palsy (PSP) (Tabaton et al., Ann. Neurol. 24: 407 (1988)), Pick""s disease (PD) (Murayama et al., Ann. Neurol. 27: 394 (1990)), Down syndrome (Papasozomenos et al., Lab Invest. 60: 123 (1989)), and frontotemporal dementias and Parkinsonism linked to chromosome 17 (FTDP-17) (Spillantini et al., Proc. Natl. Acad. Sci. USA 94: 4113 (1997)). There remains a need for the identification of effective therapies for these neurodegenerative disorders.
There is still debate as to the involvement of tau fibril formation in the onset of neurodegeneration. It is not known whether abnormal tau polymerization causes or modulates the neurodegeneration process or whether it is simply a byproduct of the process. For example, in AD it is hotly debated whether the dementia-causing pathological structures are the amyloid-beta positive senile plaques, the tau-positive neurofibrillary tangles, or a combination of both (Hardy et al., Nat. Neurosci. 1: 355 (1998)). In order to understand the etiopathogenesis of AD, there remains a need to identify molecular mechanisms which lead to the polymerization of the pathological structures themselves.
Much of what is currently known regarding tau polymerization stems from in vitro assembly assays. However, with few exceptions, the conditions that have been used to achieve tau polymerization have been extremely nonphysiological. The first experiment describing the self-association of tau protein into AD-like filaments involved 60 hours of incubation in 8M urea (Montejo de Garcini et al., J. Biochem. (Tokyo) 102: 1415 (1987)). Other experiments have required significant truncations of the molecule followed by chemical cross-linking (Wille et al., J. Cell. Biol. 118: 573 (1992)), extremely high protein concentrations (40 xcexcM) (Goedert et al., Nature 383: 550 (1996)), incubation periods up to six weeks (Schweers et al., Proc. Natl. Acad. Sci. USA 92: 8463 (1995)), or combinations of these techniques. Although relatively mild conditions have been described which result in the polymerization of low concentrations of biochemically purified tau protein (Wilson et al., J. Biol. Chem. 270: 24306 (1995)), this process can be greatly enhanced by the addition of polyanionic compounds under oxidative conditions (Goedert et al., Nature 383: 550 (1996); Kampers et al., FEBS Lett. 399: 344 (1996); Hasegawa et al., J. Biol. Chem. 272: 33118 (1997); Friedhoff et al., Biochemistry 37: 10223 (1998); Friedhoff et al., Proc. Natl. Acad. Sci. USA 95: 15712 (1998); Nacharaju et al., FEBS Lett. 447: 195 (1999)) and the addition of free fatty acids under reducing conditions (Nacharaju et al., FEBS Lett. 447: 195 (1999); Wilson et al., Am. J. Pathol. 150: 2181 (1997); King et al., Biochemistry 38: 14851 (1999); King et al., J. Neurochem. 74: 1749 (2000); Gamblin. et al., Biochemistry 39: 6136 (2000)). However, there remains a need to identify improved methods for further enhancing the polymerization of tau protein in vitro in order to help facilitate the identification of reagents which can be used to treat diseases involving tau polymerization in vivo.
Various in vitro polymerization techniques have been used to investigate the in vitro polymerization of tau. For example, it has been shown that the fatty acid induction of tau polymerization proceeds through a ligand-dependent mechanism under reducing conditions (King et al., Biochemistry 38: 14851 (1999)). Another set of experiments showed that, contrary to expectations, extensive phosphorylation of the tau molecule with various protein kinases inhibited the polyanion induction of polymerization (Schneider et al., Biochemistry 38: 3549 (1999)). Some of the factors leading to tau polymerization in the disease state are now being studied. As mentioned above, extensive tau pathology is observed in a class of neurodegenerative disorders called FTDP-17. These disease states have been linked to mutations in the tau gene that lead to missense point mutations or changes in the isoform expression of the tau protein. In vitro experiments have shown that several of the single amino acid missense point mutations found in FTDP-17 can lead to increased filament formation (Nacharaju et al., FEBS Lett. 447: 195 (1999); Gamblin et al., Biochemistry 39: 6136 (2000); Goedert et al., Nat. Med. 5: 454 (1999)). It has also been shown that tau isoforms have different polymerization characteristics, which could lead to increased tau pathology in cases of FTDP-17 with altered isoform compositions (King et al., J. Neurochem. 74: 1749 (2000)). However, a strong link between the risk factors associated with the most common neurodegenerative disorder, AD, and increased tau polymerization has not been established. Therefore, there remains a need to identify AD risk factors that are associated with tau polymerization in order to accelerate the development of effective AD therapies.
A number of risk factors have been identified which have the common characteristic of being potential contributors to oxidative stress. Thus, oxidative stress may play a major role in the etiology of Alzheimer""s disease (AD). The normal aging process, head trauma, increased levels of heavy metals (e.g., Fe, Al, Hg), and, especially in the case of AD, aggregation of the xcex2-amyloid protein (Axcex2) are all thought to be potential contributors to increased oxidative stress. In the oxidative stress hypothesis for AD, free radicals generated by these risk factors, possibly in the form of reactive oxygen species, would then attack biological molecules that are sensitive to oxidation, such as proteins, DNA, and lipids/fatty acids, causing a cascade that would eventually lead to neurodegeneration (see, e.g., Markesbery et al., Free Radic. Biol. Med. 23: 134 (1997)).
There is direct evidence that sensitive molecules in vulnerable AD brains are modified by oxidative stress. Free radicals can lead to the carbonyl derivatization of enzymes such as glutamine synthetase and creatine kinase. This process is quickly followed by protease degradation of the enzymes. DNA is also sensitive to oxidative stress. Increases in the adduct 8-hydroxy-2xe2x80x2-deoxyguanisine have been reported for mitochondrial DNA, and to a lesser extent nuclear DNA, in AD brains when compared to age-matched controls. In addition, a two-fold increase in oxidative damage to DNA through strand breaks has been described in the brains of AD patients (see, e.g., Markesbery et al., Free Radic. Biol. Med. 23:134 (1997)).
Polyunsaturated fatty acids (FA) are especially vulnerable to oxidative stress since their double bonds make the removal of H+ by free radicals relatively easy. Although some reports disagree on the location of FA oxidation in AD brain (see, e.g., Markesbery, Brain Pathol. 9: 133 (1999)), it is clear that thiobarbituric acid reactive substances (a marker for FA oxidation) are elevated in these patients. In addition, many FA breakdown products including malondialdehyde (MDA) and hydroxynonenal (HNE) can be detected at greater than normal levels in AD patients. Increased amounts of specific FA metabolites, such as the F2-isoprostanes and F4-neuroprostanes, can also be found in the affected brain regions of AD patients and even in the cerebrospinal fluid of probable AD patients (Montine et al., Neurology 52: 562 (1999)). In addition to the toxicity of some of the FA breakdown products (such as HNE), the alterations in membrane fluidity as a result of FA oxidation may also have deleterious effects in AD patients.
While it is becoming clear that oxidative stress is likely a significant contributor to the neurodegenerative process, it is not clear how these factors are related to the two major pathological structures found in AD, senile plaques and neurofibrillary tangles. Senile plaques (SPs), which consist mainly of polymerized Axcex2 protein, may contribute to oxidative stress though the generation of free radicals, but their involvement in the neurodegenerative process is not clear. While the addition of Axcex2 to cultured neurons results in increased protein oxidation and cell death (Busciglio et al., Neuron 14: 879 (1995)), animal models that contain elevated amounts of SPs do not show signs of neurodegeneration (Takeuchi et al., Am. J. Pathol. 157: 331 (2000)). In addition, the presence of SPs does not correlate well with the degree of dementia in AD patients (Arriagada et al., Neurology 42: 631 (1992)).
In contrast, neurofibrillary tangles (NFTs) consisting primarily of polymerized tau molecules do correlate well with the degree of dementia in AD (Arriagada et al., Neurology 42: 631 (1992)). In addition, an emerging class of neurodegenerative disorders that involve the fronto-temporal regions of the brain appear to be caused by pathological tau inclusions in the absence of SPs (Spillantini et al., Proc. Natl. Acad. Sci. USA 94: 4113 (1997)). Finally, the formation of tau filaments appears to directly cause neurodegeneration in an animal model. Overexpression of the tau protein in lamprey ABC neurons leads to filament formation and subsequent neuronal death (Hall et al., Proc. Natl. Acad. Sci. USA 94: 4733 (1997); Hall et al., J. Cell Sci. 113: 1373 (2000)).
Although the formation of NFTs may be relevant to the neurodegenerative process, it is not clear how they are involved with the oxidative stress hypothesis for AD. Previously, the only link between oxidative stress and tau filament formation has been the reports which describe the prerequisite oxidation of the tau molecule for its polymerization in vitro. The oxidation of a specific cysteine that results in disulfide-linked dimers of tau has been shown to be a necessary first step before the induction of tau filament formation (Schweers et al., Proc. Natl. Acad. Sci. USA 92: 8463 (1995)). It should be noted, however, that these results required special conditions to be effective. First, the experiments were performed on tau molecules truncated at both the amino- and carboxy-terminal regions so that only the microtubule binding repeat (MTBR) regions remained. Secondly, only three microtubule binding repeats could be used. This was due to the fact that there are two cysteines in the tau molecule, one in MTBR2 and one in MTBR3. If both cysteines were left in the tau constructs, they preferentially formed intramolecular disulfides instead of forming dimers.
The tau oxidation theory does not seem tenable for several reasons. The cellular markers for protein oxidation that have been identified in AD as a result of oxidative stress are the creation of protein carbonyls and the nitration of tyrosine residues (see, e.g., Markesbery et al., Brain Pathol. 9: 133 (1999)). It is not clear whether oxidative stress would actually result in the cysteine oxidation and subsequent dimerization of tau molecules. The filamentous tau structures found in AD consist of all six isoforms of the tau molecule, including those with four MTBR (see, e.g., Spillantini et al., Trends in Neurosciences, 21: 428 (1998)). Therefore, tau molecules containing two cysteines are capable of polymerizing in vivo. If cysteine oxidation of the tau molecule is a prerequisite and the intramolecular disulfide formation is favored over dimerization, one would not expect the four MTBR isoforms of tau to be present in the filaments that make up the NFTs. Therefore, there remains a need to determine the effects of oxidation on tau polymerization in vivo and the mechanism by which oxidative stress induces neurodegeneration in AD.
Using methods described in co-pending U.S. application Ser. No. 09/919,475, filed on the same date as the present application and based on U.S. Provisional Application Serial No. 60/221,777 filed on Jul. 31, 2000, specific relatively low molecular weight liqands (generally less than about 400 daltons) have been identified which inhibit and/or reverse tau filament formation or polymerization a substoichiometric concentrations relative to tau protomer. This co-pending application, which is owned by the same assignee of the present application, is hereby incorporated by reference. These liqands or inhibitors can be used therapeutically to treat certain neurological disorders or disease states in vivo, including Alzheimer""s disease, in which tau filaments are formed.
In one embodiment, the present invention provides a method for regulating the assembly of the protein tau in the brain of a patient, comprises:
identifying a patient in need of a method for inhibiting tau polymerization in the brain; and
administering to the patient a pharmacologically effective amount of an inhibitor of fatty acid oxidation, wherein the inhibitor is selected from the group consisting of (1) a first compound of the general formula 
wherein R1 is an aliphatic radical having one to six carbon atoms and wherein R2 and R3 independently are a second aliphatic radical having one to six carbon atoms, a hydroxyl-substituted aliphatic radical having one to six carbon atoms, or a pheny radical; (2) a second compound of general formula 
wherein R4, R6, and R8 are independently a third aliphatic radical having 1 to 6 carbon atoms and R5 and R7 are independently a fourth aliphatic radical having 1 to 6 carbon atoms or a second hydroxyl-substituted aliphatic radical having one to six carbon atoms; (3) a third compound of general formula 
(4) a fourth compound of general formula 
wherein R9 and R10 independently are a fourth aliphatic radical having 1 to 6 carbon atoms; (5) a fifth compound of general formula 
wherein R11 is a carboxylic acid-substituted aliphatic radical having 1 to 6 carbon atoms and R12 is a fifth aliphatic radical having 1 to 6 carbon atoms; and (6) pharmaceutically acceptable salts thereof.
Preferably, the inhibitor is the first compound of the general formula 
wherein R1 is an aliphatic radical having one to six carbon atoms and wherein R2 and R3 independently are a second aliphatic radical having one to six carbon atoms, a hydroxyl-substituted aliphatic radical having one to six carbon atoms, or a pheny radical. More preferably, the inhibitor is 2-[[4-(dimethylamino)phenyl]azo]-6-methoxylbenzothiazole (i.e., R1, R2, and R3 are methyl groups in the above formula I) having the formula Ixe2x80x2: 
In one embodiment, the patient is a human. Generally the inhibitor is administered in an effective amount which can be determined using conventional techniques. Generally, the inhibitor is administered in an amount selected from about 10 mg per day to about 1000 mg per day. In one embodiment, the administering is performed repeatedly over a period of at least one week. In one embodiment, the administering is performed repeatedly over a period of at least one month. In one embodiment, the administering is performed repeatedly over a period of at least three months. In one embodiment, the administering is performed repeatedly over a period of at least one year. In another embodiment, the administering is performed at least once monthly. In another embodiment, the administering is performed at least once weekly. In another embodiment, the administering is performed at least once daily. In another embodiment, the administering is performed at least once weekly for at least one month. In another embodiment, the administering is performed at least once per day for at least one month.